EP2089073B1 - Structures synthétiques multicouches cornéennes comprenant des fibres de collagène - Google Patents

Structures synthétiques multicouches cornéennes comprenant des fibres de collagène Download PDF

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EP2089073B1
EP2089073B1 EP07820364A EP07820364A EP2089073B1 EP 2089073 B1 EP2089073 B1 EP 2089073B1 EP 07820364 A EP07820364 A EP 07820364A EP 07820364 A EP07820364 A EP 07820364A EP 2089073 B1 EP2089073 B1 EP 2089073B1
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layer
collagen
cornea
cells
tissue
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EP2089073A1 (fr
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James Torbet
David John Stuart Hulmes
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Centre National de la Recherche Scientifique CNRS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • the invention relates to a method for the in vitro preparation of a synthetic multi-layer corneal structure comprising collagen fibers, wherein the fibers in each layer are unidirectionally and uniformly oriented, which comprises successive polymerisation of layers of a biopolymer fiber forming solution in the presence of a magnetic field, wherein the fiber orientation in at least one layer differs from that in at least one of its superior and/or inferior layer according to an angle alpha.
  • the invention also relates to a biological tissue-like cornea multi-layer structure comprising the synthetic multi-layer structure and cells inoculated therein, such as an orthogonal multi-layer collagen tissue-like cornea, and the method for its preparation.
  • the invention may be used for preventing or treating a damaged tissue, or for creating a model for biological testing, such as pharmacotoxicity testing.
  • Tissue engineering seeks to repair, replace or restore tissue function, typically by combining biomaterials and living cells.
  • the present world market for the artificial corneas is estimated to approximately 100,000 units per year, including 25,000 in Europe. Since the present sale price of an artificial corneal implant amounts to approximately 2000 euros, the market is estimated to around 120 million euros.
  • the cornea consists of three distinct cellular layers: the outer epithelium, the central stroma (composed of keratocytes embedded in a dense, highly organized extracellular matrix of collagen fibrils and proteoglycans) and inner endothelium
  • the stroma is delimited by specialized acellular structures, Bowman's and Descemet's membranes, lying at the interface between the epithelium and endothelium respectively (see Figure 7 ).
  • the stroma makes up some 90% of the corneal volume and 70% of the cornea dry weight.
  • a highly structured acellular primary stroma forms which is believed to dictate the pattern of organization of the adult stroma (Trelstad and Coulombre, 1971a).
  • the collagen fibrils composed of collagens types I and V, are of uniformly narrow ( ⁇ 30 nm) diameter and are arranged in lamellae, within which fibrils are parallel and separated by a proteoglycan rich matrix.
  • the collagen fibrils are stabilized by intra- and inter-molecular covalent crosslinks, which confer tensile strength and stabilize fibrils against proteolytic degradation. Transparency is believed to be largely dependent upon the ordered three-dimensional architecture of thin collagen fibrils.
  • the interlacing of stromal lamellae endows the cornea with highly non-linear biomechanical properties - becoming increasingly stiffer under high intra-ocular pressure which enables the cornea to survive abnormal influences such as impact, injury and surgery without bursting.
  • tissue engineered corneas should facilitate the in vitro study of the complex physiology of living tissue and would also serve as alternatives to animal models for pharmacotoxicity testing (Germain et al., 1999 ; Griffith et al., 1999 ; Builles et al, 2007). While the two in vitro human corneal models currently commercially available for the latter purpose, from SkinEthic Laboratories (Nice, France) and MatTek Corporation (EpiOcularTM; Ashland, MA, USA), provide a cornea-like epithelium, the key stromal and endothelial components are entirely absent. The validity of toxicity testing can only be enhanced by the introduction of more complete models resembling more closely normal human cornea in both composition and organization.
  • Collagen has been successfully aligned using a number of techniques. Hydrodynamic flow or electrospinning can generate oriented collagenous sheets. The former gives rise to ultrathin ribbons of highly oriented small diameter ( ⁇ 3 nm) fibrils (Jiang et al., 2004). While electrospinning results in less well oriented thicker fibers of variable diameter (Boland et al., 2004;Matthews et al., 2002). However, it has not been demonstrated that either of these techniques can give rise to anything other than uni-directionally aligned materials. Another approach exploits the spontaneous liquid crystalline ordering that takes place with time in collagen solutions at high concentration. A cholesteric phase forms in which the molecules are aligned in planes that continuously rotate in a helical manner.
  • Dense three-dimensionally ordered matrices are formed by inducing fibrillogenesis (Besseau et al., 2002;Mosser et al., 2006). The colonization of these dense collagen ( ⁇ 40 mg/ml) matrices by human dermal fibroblast to a depth of 400 ⁇ m within a month demonstrates that they have potential tissue engineering applications.
  • Highly oriented collagen (Torbet and Ronziere, 1984;Murthy, 1984) is produced when a solution of molecules is transformed into a gel in a strong magnetic field because of the cumulative effect of the many weakly diamagnetically anisotropic molecules (Worcester, 1978).
  • Many cell types including fibroblasts (Guido and Tranquillo, 1993), keratinocytes, osteoblasts (Kotani et al., 2000), neurites (Dubey et al., 1999), endothelial cells undergoing angiogenesis (Torbet et al., 2000) are aligned by contact guidance when seeded on these magnetically oriented collagen or fibrin substrates.
  • hollow tubes having an outer wall of circumferentially oriented type I collagen for bioartificial arteries (Tranquillo et al., 1996) and rods with uni-axially aligned fibrin or collagen for nerve regeneration (Ceballos et al., 1999).
  • tissue-like multi-layer structures which may be used as implants in medicine.
  • tissue-like multi-layer structures should conform the anatomy of natural tissues, such as orthogonal structure of the cornea.
  • the present Inventors have solved this problem by developing a process which comprises successive polymerisation of layers of a collagen fiber forming solution in the presence of a magnetic field.
  • a lamellar scaffold is thus obtained, comprising fibers which are unidirectionally and uniformly oriented within each layer as it can be seen in Figure 3 , resembling those present in vivo.
  • This solution offers the great advantage that the layers of oriented polymerised fibers are not altered by subsequent applications of the magnetic field, and thus keep their orientation unchanged.
  • the lamellar scaffold of the present invention may comprise a differential orientation of the collagen fibers from one layer to the next one.
  • laminated corneal stroma-like scaffolds consisting of multiple intermeshed orthogonal layers of oriented collagen fibers may be created. Keratocytes seeded on unidirectionally oriented collagen gels become uniformly oriented along the direction of the collagen fibrils and penetrate into the collagen gel where they align following the direction of the collagen fibrils within the lamellae.
  • epithelial and endothelial cells also migrate and proliferate on this scaffold they can form the basis for the reconstruction of hemi or complete cornea equivalents for use in lamellar or penetrating keratoplasty, respectively.
  • Scaffolds can be used as stand alone implants, or be seeded with cells prior to implantation or implanted after being allowed to mature in vitro until tissue-like properties develop.
  • tissue-engineered human corneal equivalents can also serve as alternatives to animal models for cosmeto-pharmacotoxicity testing.
  • three-dimensional organ mimics they can also facilitate the in vitro study of the complex physiology of living tissue.
  • the invention is aimed at a method for the in vitro preparation of a synthetic multi-layer corneal structure comprising or consisting of collagen fibers, wherein the fibers in each layer are unidirectionally and uniformly oriented, which comprises the following steps:
  • multi-layer refers to a structure comprising two or more layers.
  • oriented or “unidirectionally oriented” refers herein to biopolymer fibers which are substantially aligned in a specific direction. This orientation can be checked using birefringence and/or electronic microscopy and/or optical microscopy.
  • uniformly means that the orientation is uniform throughout a given lamellae, without any patches of random fiber alignment.
  • the holder refers to any container which is appropriate for use in the present invention, in particular for allowing the biopolymer to orient upon application of the magnetic field. It may be of any shape, such as a well having a flat base, or a mold, such as a cylindrical mold having an annular cross-section.
  • the cylindrical mold can be positioned vertically in a horizontal magnetic field.
  • Such cylindrical molds having an annular cross-section find an application for preparing tissue-like compact bones or intervertebral discs.
  • the well having a flat base can be positioned horizontally in a horizontal magnetic field.
  • Wells are preferably used for preparing tissue-like corneas.
  • the holder has an impermeable or semi-permeable base.
  • the fiber orientation in each layer differs from that in at least one of its superior and/or inferior layer according to the angle alpha.
  • the collagen is in the form of a solution in certain conditions, and can form fibers by modifying said conditions.
  • the method of the invention also requires that the collagens orient in a unique direction upon the application of a magnetic field.
  • the collagen fibers are type I and/or type V collagen fibers, and/or any other fiber forming collagen.
  • the thickness of the layers of the biopolymer fiber forming solution in the holder is advantageously less than 3 mm.
  • the collagen fiber forming solution is acid and cold, and that collagen fibers are obtained by raising the pH and ionic strength of acid-soluble collagen as well as by heat precipitation of neutral salt-soluble collagen (see for example Murthy, 1984).
  • the collagen fiber formation is thus induced by neutralising and/or heating the collagen fiber forming solution, advantageously to a pH ranging from 7 to 8 and/or to a temperature ranging from 28°C to 35°C, advantageously 30°C.
  • the concentration of the collagen fiber forming solution ranges from 1 to 10 mg/mL.
  • the neutralisation of the solution is made prior to the heating step, thus allowing to introduce the neutralised biopolymer fiber forming solution in the holder to form one layer, and the heating step is made in the presence of the magnetic field, in order to obtain fibers which are unidirectionally oriented in the layer.
  • the physical and/or biological properties of the synthetic multi-layer structure may be changed by adding compounds, preferably biocompatible compounds.
  • proteoglycans in solution may be added for improving optical transparency of the synthetic multi-layer structure.
  • the synthetic multi-layer structure further comprises proteoglycans. More advantageously, said proteoglycans are added before fiber formation. Most advantageously, said proteoglycans are added to the neutralised biopolymer fiber forming solution.
  • covalent cross-linkages are introduced in order to confer tensile strength and stabilize fibers against proteolytic degradation. Introduction is advantageously made to the obtained synthetic multi-layer structure.
  • the synthetic multi-layer structure further comprises covalent cross-linkages obtained using chemical compounds such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS).
  • chemical compounds such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS).
  • the synthetic multi-layer structure further comprises covalent cross-linkages obtained using enzymes such as transglutaminase or lysyl oxidase.
  • the collagen fibers to be used in the invention may result from an extract of a biological tissue, or may be recombinant.
  • the collagen fibers are recombinant collagen fibers, advantagesouly human recombinant collagen fibers.
  • Recombinant collagen fibers may be obtained from FIBROGEN Inc.
  • the synthetic multi-layer structure is dehydrated by evaporation or blotting using a semi-permeable support. Dehydratation allows to concentrate the synthetic multi-layer structure in order to obtain a structure which resembles those present in vivo.
  • the concentration of the synthetic multi-layer structure ranges from 100 mg/mL to 200 mg/mL, advantageously 160 mg/mL.
  • the synthetic multi-layer structure is lyophilized.
  • Lyophilized synthetic multi-layer structures of the invention are very appropriate for storage. Lyophilization further allows to render the structure more porous.
  • the appropriate magnetic field for use in the present invention is generally a high strength (4.7 to 9 Tesla) magnetic field.
  • the magnetic field is a static magnetic field, and may be generated by a horizontal bore electromagnet, such as those from the Oxford Company of the United Kingdom.
  • the angle alpha is comprised between 30° and 150°. In a particularly advantageous embodiment, the angle alpha is about 90°, thus obtaining a synthetic orthogonal multi-layer structure.
  • the invention is aimed at a synthetic multi-layer corneal structure comprising or consisting of collagen fibers, wherein the collagen fibers in each layer are unidirectionally and uniformly oriented, said structure being obtainable by the method for the in vitro preparation of a synthetic multi-layer corneal structure according to the invention.
  • the fiber orientation in at least one layer differs from that in at least one of its superior and/or inferior layer according to the angle alpha.
  • the collagen fibers are type I and/or type V collagen fibers.
  • the synthetic multi-layer structure further comprises proteoglycans.
  • the synthetic multi-layer structure further comprises covalent cross-linkages obtained using chemical compounds such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS).
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide ester
  • the synthetic multi-layer structure further comprises covalent cross-linkages obtained using enzymes such as transglutaminase or lysyl oxidase.
  • the collagen fibers are recombinant collagen fibers, advantageously human recombinant collagen fibers.
  • the synthetic multi-layer structure according to the invention is lyophilized.
  • the angle alpha is comprised between 30° and 150°. More preferably, the angle alpha is 90°, and the structure is a synthetic orthogonal multi-layer structure.
  • the invention is directed to a method for the in vitro preparation of a biological tissue-like cornea multi-layer structure which comprises the preparation of a synthetic multi-layer corneal structure according to the method of the present invention, and the inoculation of cells therein.
  • the cells to be inoculated may be of any type.
  • the cell type is generally that of the tissue which it is intended to re-create, for example smooth muscle cells for a muscle-like tissue.
  • the cells are mammalian cells, more preferably human cells.
  • the multi-layer collagen tissue-like cornea structure is a corneal stroma, hemi-cornea or complete cornea.
  • the cells are keratocytes, endothelial cells, epithelial cells or a combination thereof.
  • Cells may be added to the biopolymer fiber forming solution prior to assembly of the fibers and following adjustment to near physiological conditions of pH and ionic strength. Cells may also be added to the previously formed multi-layer structure. Thus, in a preferred embodiment, the cell inoculation is made by adding the cells to the neutralised collagen fiber forming solution, and/or by adding the cells to the multi-layer collagen tissue-like structure.
  • the invention is aimed at a biological tissue-like multi-layer corneal structure comprising the synthetic multi-layer corneal structure according to the invention and cells inoculated therein.
  • Said biological tissue-like multi-layer corneal structure may be obtained by the method as described above.
  • the cells are mammalian cells, advantageously human cells.
  • the biological tissue-like multi-layer structure according to the invention is a multi-layer collagen tissue-like corneal structure, advantageously an orthogonal multi-layer collagen tissue-like cornea, such as a corneal stroma, a hemi-cornea or a complete cornea.
  • the cells are keratocytes, endothelial cells, epithelial cells or a combination thereof.
  • the biological tissue-like multi-layer structure may further resemble any tissue having a multi-layer structure, such as those where the direction alternates from one layer to the next, including, for example, in addition to cornea, compact bone or intervertebral disc.
  • the invention is aimed at the use of the synthetic multi-layer structure according to the invention for the preparation of a biological tissue-like multi-layer structure.
  • the present invention is aimed at the biological tissue-like multi-layer corneal structure according to the invention for use as a medicament.
  • the invention is aimed at the use of the biological tissue-like multi-layer corneal structure according to the invention for the preparation of a medicament for preventing or treating a damaged biological tissue in a subject, such as a mammal, preferably a human.
  • Also comprised herein is a method of treating and/or preventing a damaged biological tissue in a subject using the biological tissue-like multi-layer corneal structure according to the invention.
  • the present invention is aimed at the use of the biological tissue-like multi-layer corneal structure according to the invention as a model for biological testing, such as pharmacotoxicity testing.
  • the invention is directed to the use of the orthogonal multi-layer collagen tissue-like cornea according to the invention for the preparation of a medicament for preventing or treating a damaged corneal tissue in a subject, such as corneal stroma, hemi-cornea, complete cornea or a combination thereof.
  • the invention also relates to the use of the orthogonal multi-layer collagen tissue-like cornea according to the invention for treating a damaged corneal tissue in a subject, such as corneal stroma, hemi-cornea, complete cornea or a combination thereof.
  • the cells are keratocytes, endothelial cells, epithelial cells or a combination thereof.
  • Also comprised herein is a method of treating and/or preventing a damaged corneal tissue in a subject using the orthogonal multi-layer collagen tissue-like cornea according to the invention.
  • Sample holders were either glass bottomed plastic culture dishes (MatTek Co.) or Transwell® semi-permeable polycarbonate supports (Coming Co.).
  • the culture dishes (outer diameter 35 mm) had a shallow (1.5 mm deep) central glass well (14 mm diameter).
  • Transwell plates were cut to produce individual wells.
  • the wells (12 mm inner diameter) were placed in standard 35 mm plastic petri dishes.
  • the pore diameter of the semi-permeable membrane was 0.4, 3 or 12 ⁇ m.
  • the volume of neutralized collagen solution added to the wells varied from 80 to 200 ⁇ l which correspond to a fully hydrated thickness of 0.5 to 1.3 mm. The thickness of the gel is controlled by the volume of the initial solution.
  • a split-coil superconducting magnet (Thor Cryogenics) with horizontal room temperature bore was used.
  • the bore was horizontal, 5 cm diameter and had a total length of 40 cm.
  • a temperature stabilizing jacket reduces the effective diameter to about 4.5 cm.
  • the field profile rose to a flat plateau extending ⁇ 5 cm about the bore centre making it possible to simultaneously place up to three 35 mm diameter samples in maximum field.
  • the temperature of the sample space was controlled by circulating water from a temperature controlled bath.
  • the field used for processing was near the maximum possible (7 tesla). This magnet was provided by the Grenoble High Magnetic Field Laboratory funded by the CNRS.
  • Turbidity defined as the optical density, was measured at 400 nm using a Beckman spectrophotometer. Solutions were mixed in the cold and transferred to a prewarmed sample cell (1 mm optical path length quartz cells) positioned in the spectrophotometer.
  • Dehydrated scaffolds could be conserved for several months but once rehydrated with conservation medium they were used within 7 days.
  • the conservation medium was 200 ⁇ l of DMEM using double strength antibiotic concentration at +4°C DMEM 50 ml; Penicilline G 50 ⁇ l (200 UI/ml); Gentamycin 100 ⁇ l (40 ⁇ g/ml); Fungizone 100 ⁇ l (2 ⁇ g/ml)
  • Keratocytes were isolated from human corneas removed in accordance with ethical regulations. The corneas were strored at 31°C in organ culture, but were unusable for treatment because their endothelial density was too low. Isolation was performed with collagenase A (Roche, ref. 10103586), 3 mg/ml for 3 hours at 31 °C with stirring at 200 rpm. The digest was purified through a 70 ⁇ m cell sieve (BD Falcon ref. 352350) and immediately placed in monolayer culture.
  • Keratocytes were seeded at a density of 10,000 cells/cm 2 then cultured in a specially designed medium providing optimal growth and preservation of the phenotype as followed : DMEM/Ham-F12 1:1, 10% NCS, 5 ng/ml bFGF, antibiotics. The medium was changed every two days until cell confluence was reached. At confluence, cells were resuspended by trypsin-EDTA 0.5g trypsin/1 and 0.2 g EDTA/1 (Sigma ref. CR0303), and amplified over 3 passages (from P0 to P2) and seeded at P3 inside the matrix. SEL, TEM methods ...
  • FIG. 1 A typical type I collagen assembly curve obtained following neutralization of acid soluble collagen with buffer is shown in figure 1 .
  • the resulting gel (2mg/ml, 1mm thick) is translucent.
  • an aligned gel is produced ( fig. 2A ).
  • collagen molecules orient perpendicular to the applied field direction due to the negative value of their diamagnetic anisotropy (Worcester, 1978c), high alignment results thanks to the influence of surface constraints (Torbet and Ronziere, 1984a).
  • the fibrils are two to four times thicker (60-110 nm) than those of native stroma and they are also frequently aggregated into large bundles which are generally better oriented than single fibrils.
  • the degree of orientation is sufficient to induce contact guidance of keratocytes as shown below.
  • the preparative procedures required for SEM visualization significantly compact and possibly distort the gel.
  • the final concentration of collagen in the oriented gels ( ⁇ 2 mg/ml) is well below that present in cornea stroma, which is in the region of 160 mg/ml.
  • the hydrated scaffolds can be readily concentrated into sheets to any desired degree with little or no loss in orientation either by evaporation or blotting the semipermeable support. Evaporation is slow and results in the build-up of salt and buffer, while blotting is rapid and causes no change in osmotic conditions, consequently it can be used in the presence of cells.
  • the fully hydrated scaffolds have also been lyophilized to provide porous sponges but so far we have not carried out cell growth studies on this support.
  • Figure 6 It is not surprising in the light of passed results that keratocytes align on the surface of oriented collagen ( fig. 5 ). From the point of view of cornea reconstruction it is vital that cells penetrate into the stroma-like scaffold and continue to be steered by contact guidance.
  • Figure 6 shows an example of a three orthogonal lamellae supported on a semi-permeable membrane and seeded with keratocytes. It is clear from this image that the cells both penetrate the matrix and align along the direction of collagen constituting each lamella.
  • the Inventors have also shown that the growth of epithelial and endothelial cells is also supported by this matrix.
  • an orthogonal lamellar collagen-based scaffold which supports oriented keratocyte growth and penetration in vitro.
  • This scaffold can form the basis of an implant that will promote regeneration of damaged cornea in such a way as to give rise to the essential properties inherent to native stroma.
  • An acellular collagenous matrix of orthogonal lamellae constitutes the primary stroma that serves as the template on which the adult cell-infiltrated stroma organizes (Trelstad and Coulombre, 1971b). It has also been shown (Hu et al., 2005) that the properties of the corneal stroma reconstructed following grafting of an artificial matrix resemble (optical clarity, fibril diameter) those of native stroma.
  • scaffolds of stacked lamellae of oriented fibrin can be created in the way described here for collagen.
  • Laminated collagen-fibrin composites can also be produced in which alternate layers have different physical and bioactive properties.
  • Lamellae composed of fibrin or collagen-fibrin have also the potential to be useful in cornea medication.
  • Unoriented fibrin is already used as a support for human cornea-limbal epithelial cells, which are grafted on to the damaged corneal surface (Rama et al., 2001;Pellegrini et al., 1997). This application might be improved by using oriented single sheets or orthogonal multilayers which would more closely mimic the structure of the underlying stroma.
  • crosslinking The above studies were carried out on uncrosslinked collagen. Scaffolds can be crosslinked chemically or using enzymes such as transglutaminase or lysyl oxidase. The latter can probably be used in the presence of cells if desirable. Crosslinking stiffens the scaffold making it easier to manipulate and also alter the rate of matrix degradation.
  • the human stroma contains about 200 lamellae each about 2 ⁇ m thick composed of 160 mg/ml collagen.
  • the Inventors have developed a novel procedure for the production of oriented collagen based scaffolds for use in tissue engineering.
  • the procedure results in a layered structure, within each layer the collagen fibrils are oriented uniaxially while the angle between the fibrils in each successive layer can be chosen at will.
  • Such a twisted plywood structure is seen in a number of connective tissues (for example, cornea, compact bone, intervertebral disc).
  • Collagen based sponges and sheets which might have other applications in the reconstruction of tendon and muscle, have also been produced. Keratocyte alignment is directed by the collagen orientation both on the surface and within the bulk of the scaffold. All three corneal cell types grow on this scaffold.
  • the scaffold thus has the potential to act as a stand alone matrix or provide the basis for more sophisticated cell supporting constructs.

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Claims (20)

  1. Procédé pour la préparation in vitro d'une structure synthétique multicouche cornéenne comprenant des fibres de collagène, dans lequel lesdites fibres dans chaque couche sont orientées unidirectionnellement et uniformément, qui comprend les étapes suivantes consistant à :
    a) positionner un support contenant une couche de solution pour former des fibres de collagène dans un champ magnétique et à induire la formation des fibres en neutralisant et en chauffant la solution pour former des fibres de collagène afin d'obtenir des fibres qui sont orientées unidirectionnellement et uniformément dans la couche,
    dans lequel on neutralise la solution avant l'étape de chauffage, ce qui permet ainsi d'introduire la solution pour former des fibres de collagène dans le support afin de former une couche, et l'étape de chauffage est effectuée en présence du champ magnétique afin d'obtenir des fibres orientées unidirectionnellement dans la couche,
    b) introduire une autre couche de solution pour former des fibres de collagène dans le support, ladite autre couche étant introduite sur la couche obtenue à l'étape a),
    c) positionner le support dans le champ magnétique et induire la formation des fibres en neutralisant et en chauffant la solution pour former des fibres de collagène afin d'obtenir des fibres qui sont orientées unidirectionnellement et uniformément dans ladite autre couche,
    dans lequel la neutralisation de la solution est effectuée avant l'étape de chauffage, ce qui permet ainsi d'introduire la solution pour former des fibres de collagène neutralisée dans le support afin de former une couche et l'étape de chauffage est effectuée en présence du champ magnétique afin d'obtenir des fibres qui sont orientées unidirectionnellement dans la couche,
    d) répéter facultativement les étapes précédentes, et ainsi obtenir une structure synthétique multicouche cornéenne, dans lequel l'orientation des fibres dans au moins une couche diffère de celle dans au moins l'une de ses couches, supérieure et/ou inférieure, selon un angle alpha, et où ladite orientation différentielle entraîne le changement, par une rotation selon l'angle alpha, de la position du support de l'étape c) par rapport à la position du support de l'étape a) .
  2. Procédé selon la revendication 1, dans lequel l'orientation des fibres dans chaque couche diffère de celle dans au moins l'une de ses couches, supérieure et/ou inférieure, selon l'angle alpha.
  3. Procédé selon la revendication 1 ou 2, dans lequel la structure synthétique multicouche cornéenne comprend en outre des protéoglycanes.
  4. Procédé selon l'une quelconque des revendications 1 à 3, dans lequel la structure synthétique multicouche cornéenne comprend en outre des réticulations covalentes obtenues en utilisant des composés chimiques tels que le chlorhydrate de 1-éthyl-3-(3-diméthylaminopropyl)carbodiimide (EDC) ou l'ester de N-hydroxysuccinimide (NHS).
  5. Procédé selon l'une quelconque des revendications 1 à 4, dans lequel la structure synthétique multicouche cornéenne comprend en outre des réticulations covalentes obtenues en utilisant des enzymes telles que la transglutaminase ou la lysyl oxydase.
  6. Procédé selon l'une quelconque des revendications 1 à 5, dans lequel la structure synthétique multicouche cornéenne est déshydratée par évaporation ou par absorption en employant un support semi-perméable.
  7. Procédé selon l'une quelconque des revendications 1 à 6, dans lequel la structure synthétique multicouche cornéenne est lyophilisée.
  8. Procédé selon l'une quelconque des revendications 1 à 7, dans lequel l'angle alpha est de 90°, pour ainsi obtenir une structure synthétique multicouche cornéenne orthogonale.
  9. Structure synthétique multicouche cornéenne comprenant des fibres de collagène, dans laquelle lesdites fibres dans chaque couche sont orientées unidirectionnellement et uniformément et dans laquelle l'orientation des fibres dans au moins une couche diffère de celle dans au moins l'une de ses couches, supérieure et/ou inférieure, selon un angle alpha, ladite structure pouvant être obtenue par le procédé selon l'une quelconque des revendications 1 à 8.
  10. Procédé pour la préparation in vitro d'une cornée de type tissulaire à base de collagène multicouche qui comprend la préparation d'une structure synthétique multicouche cornéenne selon le procédé selon l'une quelconque des revendications 1 à 8 et l'inoculation de cellules à l'intérieur de celle-ci.
  11. Procédé selon la revendication 10, dans lequel ladite cornée de type tissulaire à base de collagène multicouche est le stroma cornéen, l'hémicornée ou la cornée complète.
  12. Procédé selon la revendication 10 ou 11, dans lequel les cellules sont les kératocytes, les cellules endothéliales, les cellules épithéliales ou une combinaison de ceux-ci.
  13. Procédé selon l'une quelconque des revendications 10 à 12, dans lequel l'inoculation des cellules est effectuée en ajoutant les cellules à la solution formant les fibres de collagène neutralisée et/ou en ajoutant les cellules à la cornée de type tissulaire à base de collagène multicouche.
  14. Cornée de type tissulaire à base de collagène multicouche comprenant la structure synthétique multicouche cornéenne selon la revendication 9 et les cellules inoculées dans celle-ci.
  15. Cornée de type tissulaire à base de collagène multicouche selon la revendication 14, qui est une cornée de type tissulaire à base de collagène multicouche orthogonale, par exemple le stroma cornéen, l'hémicornée ou la cornée complète.
  16. Cornée de type tissulaire à base de collagène multicouche selon la revendication 15, dans laquelle les cellules sont les kératocytes, les cellules endothéliales, les cellules épithéliales ou une combinaison de ceux-ci.
  17. Utilisation de la structure synthétique multicouche cornéenne selon la revendication 9, pour la préparation d'une cornée de type tissulaire à base de collagène multicouche.
  18. Cornée de type tissulaire à base de collagène multicouche selon l'une quelconque des revendications 14 à 16 destinée à être utilisée comme médicament.
  19. Utilisation de la cornée de type tissulaire à base de collagène multicouche selon l'une quelconque des revendications 14 à 16 comme modèle pour effectuer des tests biologiques, par exemple des tests de pharmacotoxicité.
  20. Utilisation de la cornée de type tissulaire à base de collagène multicouche orthogonale selon la revendication 15 ou 16 pour la préparation d'un médicament destiné à prévenir ou à traiter le tissu cornéen abîmé d'un sujet, par exemple le stroma cornéen, l'hémicornée, la cornée complète ou une combinaison de ceux-ci.
EP07820364A 2006-09-20 2007-09-19 Structures synthétiques multicouches cornéennes comprenant des fibres de collagène Not-in-force EP2089073B1 (fr)

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PCT/EP2007/059920 WO2008034854A1 (fr) 2006-09-20 2007-09-19 Structures multicouches synthétiques dotées de fibres biopolymères

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ATE523212T1 (de) 2011-09-15
WO2008034854A1 (fr) 2008-03-27
JP2010504122A (ja) 2010-02-12
ES2373124T3 (es) 2012-01-31

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